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Updated: May 9, 2026

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes
Published on: May 25, 2018
Sequence-specific inhibition of a nonspecific protease
Leigh A Logsdon1, Adam R Urbach
1Department of Chemistry, Trinity University, 1 Trinity Place, San Antonio, Texas 78212, USA.
Abstract:
A nonspecific exopeptidase, aminopeptidase N (APN), is inhibited sequence-specifically by a synthetic host, cucurbit[7]uril (Q7), which binds with high affinity and specificity to N-terminal phenylalanine (Phe) and 4-(aminomethyl)phenylalanine (AMPhe) and prevents their removal from the peptide. Liquid chromatography experiments demonstrated that in the presence of excess Q7, APN quantitatively converts the pentapeptides Thr-Gly-Ala-X-Met into the dipeptides X-Met (X = Phe, AMPhe). The resulting Q7-bound products are completely stable to proteolytic digestion for at least 24 h. Structure-activity studies revealed a direct correlation between the extent of protection of an N-terminal amino acid and its affinity for Q7. Therefore, Q7 provides predictable sequence-specificity to an otherwise nonspecific protease and enables the production of a single peptide product. Conversely, APN uncovers a high-affinity epitope that is subsequently bound by Q7, and thus this approach should also facilitate the molecular recognition of peptides.
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